Three new Melanoleuca species (Agaricales, Basidiomycota) from north-eastern China, supported by morphological and molecular data

Abstract Three new Melanoleuca species, M. chifengense, M. griseoflava and M. pallidorosea, were discovered in the northeast of China. Melanoleuca chifengense is morphologically characterised by its grey to yellowish-grey pileus, decurrent lamellae, grey to yellowish-brown stipe, yellowish-grey context, ellipsoid basidiospores with irregular warts and lack of hymenial cystidia. Melanoleuca griseoflava is mainly characterised by its greyish-brown pileus, adnexed to adnate lamellae, greyish-yellow context, fusiform cystidia and almost reticulate basidiospores. Melanoleuca pallidorosea is characterised by its pinkish-white pileus, white and decurrent lamellae, ellipsoid basidiospores with round and scattered warts and lack of hymenial cystidia. The phylogenetic relationship of the three species was determined by the analyses of the ITS region and the combined data matrix (ITS-nrLSU-RPB2), respectively. The results showed that the three species formed three independent lineages. Based on the combination of both morphological and molecular data, M. chifengense, M. griseoflava and M. pallidorosea were confirmed to be new species. The morphological similarities of the three new species is also discussed.


Introduction
Melaleuca Pat. was erected by Patouillard in 1887. As the name 'Melaleuca' was found to be the same as that of a plant species, Patouillard (1897) changed it to the current name Melanoleuca Pat. The genus was traditionally included in the family Tricholomataceae, subtribus Leucopaxillaceae Singer mainly because the species present a regular hymenophoral trama, amyloid basidiospores and a white spore print (Singer 1948;Singer 1986). However, molecular data showed that the genus Melanoleuca is close to the species of Pluteaceae and Amanitaceae (Moncalvo et al. 2002;Bodensteiner et al. 2004;Matheny et al. 2006;Garnica et al. 2007;Justo et al. 2011;Vizzini et al. 2011;Binder et al. 2014;Yu et al. 2014). Therefore, Melanoleuca was assumed to belong to the Pluteoid clade by Matheny et al. (2006) and Sánchez-García et al. (2014).
The species of Melanoleuca are often characterised by having a convex to slightly depressed pileus, mostly hymenial cystidia, amyloid ornamented basidiospores and all hyphae without clamp connections (Singer 1986;Boekhout 1988;Vizzini et al. 2011). The genus Melanoleuca always grows directly on humus-rich soil, in meadows, in and outside of woods and is distributed in temperate and frigid zones of both hemispheres (Singer 1986). In recent years, many new species of Melanoleuca have been reported around the world (Vizzini et al. 2010Sánchez-García et al. 2013;Antonín et al. 2014Yu et al. 2014;Nawaz et al. 2017;Xu et al. 2019;Antonín et al. 2021). Up to now, there are 221 validly published names reported in the world (Index Fungorum 2021).
Although Melanoleuca has been proved to be a monophyletic group, the classification system within the genus remains controversial. Based on the colour of the pileus and the size of the carpophore, Singer (1986) divided the genus into four sections, i.e. sect. Alboflavidae Singer, sect. Humiles Singer, sect. Oreinae Singer and sect. Melanoleuca Pat. As Boekhout (1988) believed that the cystidia should play an important role in the classification system of Melanoleuca, the genus was, therefore, divided into three subgenera, based on the types of cystidia, i.e. subgen. Macrocystis Boekhout, subgen. Melanoleuca Pat. and subgen. Urticocystis Boekhout. Subgen. Macrocystis and subgen. Urticocystis are characterised by the presence of fusiform to lageniform cystidia and urticiform cystidia, respectively while subgen. Melanoleuca is characterised by the absence of cystidia. However, these morphological classification systems are not supported by molecular data. The result of ITS region analysis supported the fact that Melanoleuca included two subgenera, i.e. subgen. Urticocystis and subgen. Melanoleuca . The species of subgen. Melanoleuca are characterised by basidiomata with non-septate macrocystidia. Subgen. Urticocystis was composed of the taxa mainly with urticocystidia, but also without any cystidia and with macrocystidia and brightly coloured pilei .
In this paper, the authors studied three Melanoleuca species collected in north-eastern China from 2017 to 2019. Morphological observation and phylogenetic analyses confirmed that they are novel taxa in the genus Melanoleuca.

Morphological studies
All of the fungal specimens were described and photographed in the field. Specimens were dried in an electric drier and deposited in the Fungal Herbarium of Shenyang Agricultural University (SYAU-FUNGI) and Fungal Herbarium of Chifeng University (CFSZ). Tissue blocks were removed from the inner part of the fresh basidiomata for DNA analyses. Macroscopic characters of the basidiomata described here were based on observations of fresh specimens. The names of colours were based on Kornerup and Wanscher (1963). Methods used for morphological descriptions followed those of Li et al. (2017). For the microscopic study, dried materials were observed in 5% potassium hydroxide (KOH) solution. Melzer's reagent was used for testing colour reactions of the tissues and basidiospores. The notation "(n/m/p)" of basidiospores indicates that the measurements were conducted for n basidiospores from m basidiomata of p collections. The Q value (length:breadth ratio) for each spore was calculated and the mean values are presented in the descriptions. For observation of the surface of the spores, the gills were covered with a thin gold film by using an Ion Sputter Coater (MC1000, Hitachi, Japan) before imaging by a scanning electron microscope (REGLUS 8100, HITACHI, Japan). Line drawings were prepared by freehand.

DNA extraction, PCR amplification and sequencing
Total genomic DNA was extracted from fresh blocks of tissue, dried with silica gel using the cetyltrimethylammonium bromide (CTAB) method (Doyle and Doyle 1987). Primer pairs ITS5/ITS4 (White et al. 1990), LR0R/LR5 (Michot et al. 1984) and b6F/b7.1R (Matheny et al. 2007) were used to amplify the internal transcribed spacer (ITS) region, the large subunit nuclear ribosomal RNA (nrLSU) region and the second largest subunit of the nuclear RNA polymerase enzyme II (RPB2), respectively. PCR protocol and sequencing were conducted as described by Wang et al. (2019).

Phylogenetic analyses
High-quality and representative sequences of Melanoleuca in previous studies (Sánchez-García et al. 2013;Yu et al. 2014;Antonín et al. 2014Antonín et al. , 2015Antonín et al. , 2017Nawaz et al. 2017;Xu et al. 2019;Antonín et al. 2021) were downloaded from GenBank and aligned with the sequences obtained from this study by Bioedit v7.0.9 (Hall 1999) and MAFFT v7.313 (Katoh and Standley 2013). Pluteus romellii (AY854065 for ITS; AY634279 for nrLSU; AY786063 for RPB2) was used as the outgroup in this study. Data partition homogeneity tests (Farris et al. 1995) were implemented in PAUP 4.0b4a (Swofford 2003). This test detected no conflicts among ITS, nrLSU and RPB2 regions (P-value = 0.33), suggesting that sequences of the three genes can be combined for phyloge-netic analysis. The final ITS data matrices consisted of 125 samples of 669 characters, whereas the combined data set (ITS-nrLSU-RPB2) consisted of 67 samples of 2204 characters. Maximum likelihood (ML) analysis was performed with RAxML-8.2.10-WIN using a GTR-GAMMA model of evolution (Stamatakis 2014). Nodal bootstrap support (BS) was assessed with nonparametric bootstrapping using 1000 replicates. Bayesian Inference (BI) analysis was conducted with MrBayes v.3.2.6 (Ronquist et al. 2012). ModelFinder (Kalyaanamoorthy et al. 2017) and PartitionFinder 2 (Lanfear et al. 2016) were used for the selection of the best-fitting model of sequence evolution for ITS dataset (GTR+I+G+F) and the combined dataset (GTR+I+G for ITS and nrLSU, SYM+I+G for RPB2), respectively. Both of the two data sets were run for 5 000 000 generations, with four chains, and trees sampled every 500 generations. The average split frequencies were checked to determine optimal convergence of the chains below 0.01. The first 25% of the sample trees was designated as burn-in, and the remaining samples were retained for further analyses. The topologies were used to generate a 50% majority-rule consensus tree for posterior probabilities (PP). The best tree was viewed in FIGTREE v1.4.4 (Rambaut 2018) and was compiled in Adobe Illustrator CC. Both of the final alignments were submitted to TreeBASE (Submission ID 28200).

Molecular phylogenetic results
The GenBank accession numbers of the sequences, determined in this study, are from MW258676 to MW258689 and MW281543 to MW281548 (Table 1). The BI and ML analyses produced similar topologies for the ITS and combined regions datasets. The BI trees were selected for display (Figures 1, 2). The results showed that the species in the genus Melanoleuca formed a monophyletic group in both ITS regions and combined regions analyses (PP=1.00, BS=100, Figures 1, 2), which is consistent with the previous results (Yu et al. 2014;Vizzini et al. 2011). A total of five clades (A to E) can be recognized within Melanoleuca (Figures 1, 2). Based on the analyses of the two datasets, the collections named M. grisoflava (SYAU-FUNGI-061 to SYAU-FUNGI-064) formed an independent lineage with strong statistical support (PP =   Description. Pileus 30-60 mm diam., flat at first, becoming depressed at disc when mature, margin sometimes cracking, surface glabrous, grey to yellowish-grey (4B1 to 4B2), greyish-brown (4B4 to 4B6) at centre, often darker at margin. Lamellae crowded, adnate to decurrent, white to yellowish-white (4A2), 2.5-3.0 mm broad, with lamellulae, edge entire. Stipe cylindrical, 20-35 mm long × 2-5 mm diam., central, broadened at base, solid, surface grey to yellowish-grey at first (4B1 to 4B2), becoming yellowish-brown (5D8, 5E8) with age or after touching, striate, often with whitish basal tomentum. Pileus context up to 10 mm thick near stipe attachment, thin at margin, yellowish-grey (4B2), grayish brown to yellowish brown (5D3 to 5E5) in stipe cortex, up to brown (6E7) in stipe base. Odour none, taste mild. Spore print white.
Habit, ecology and distribution. On soil or meadow outside of a forest, often on the roadside near a forest. Known from north-eastern China.
In the present study, both phylogenetic analyses, based on a single region (ITS) and three regions (ITS-nrLSU-RPB2), showed that there were nine clades in the genus Melanoleuca (Figures 1, 2). According to the phylogram, M. griseoflava is sister to the other four species in clade A, i.e. M. arcuata, M. heterocystidiosa, M. robusta and M. sub-pulverulenta. Melanoleuca arcuata differs by its brick-red pileus and decurrent lamellae (Fries 1821). The other two species, M. heterocystidiosa and M. subpulverulenta, can also be easily separated from M. griseoflava, based on their small basidiomata (Singer 1939;Bon 1984); M. robusta differs on account of its grey-brown pileus, grey lamellae, brown context and caespitose growth . In clade E, M. chifengense is closely related to M. humilis and M. malenconii with high support. However, both the two species differ from M. chifengense in their dark brown pileus (Fries 1821;Bon 1990). In the analysis of both ITS region and three regions (ITS-nrLSU-RPB2), Melanoleuca pallidorosea form an individual clade (clade I) and far away from the other species of Melanoleuca.